This proposal is the competitive renewal of grant HL62969. Our studies center on nitric oxide (NO) and its interactions with heme proteins, using a variety of protein examples and numerous, largely biophysical, approaches. NO regulates activities in all mammalian tissues and plays a central role in diverse areas, including vasodUation, blood coagulation, memory formation, oxygen stress, apoptosis, and macrophage killing of invading species. NO is produced by NO synthase and detected by soluble guanylate cyclase (sGC), both of which interact with NO through heme. Other molecules possibly influenced by NO include cytochrome oxidase, hemoglobin, and gene regulatory proteins with free thiols. We have entered this field through a family of insect herne proteins, called nitrophorins, that transport NO from the saliva of the insect to the tissues of potential victims, leading to vasodilation, reduced blood coagulation and a satisfying blood meal for the insect. We have expressed and characterized five nitrophorins, four from Rhodnius prolixus, the kissing bug, which transports the trypanosome for Chagas' disease, and one from Cimex lectularius, the bed bug. We have determined crystal structures of four nitrophodns, to as high as 0.85 A resolution, and have kinetic, spectroscopic, thermodynamic, mutagenic, theoretical and computational studies underway to link structure to function in NO transport. Thus studies have revealed heme molecules that are highly distorted (ruffled), which may serve to stabilize the ferric oxidation state required for nitrophorin function. They have also revealed a substantial NO-induced conformational change in the nitrophorins that apparently serves to desolvate and protect NO from reaction with water and oxygen. We are also extending our studies to other NO sensing proteins, including sGC. In the next funding period, we propose experiments to (1) understand the role of heme distortion in nitrosyl complex formation and signaling; (2) understand the role of protein conformational change in NO binding and release by the Rhodnius nitrophorins; (3) uncover the mechanism for NO transport by Cimex nitrophorin, which appears to involve reversible S-nitrosylation; and (4) produce and characterize recombinant sGC, a complicated protein of central importance to NO signaling. These studies will provide unprecedented insight into the complicated chemistry of NO signaling.